Our laboratory recently published a report demonstrating the ability of integration defective lentiviral vectors (IDLV's) to cure hemophilia B in preclinical settings, providing additional proof of their therapeutic potential. However, further improvements of the IDLV gene delivery system are required in order to establish IDLV's as a therapeutic modality for nonfatal human diseases such as hemophilia B. Thus, we propose: a) to employ a novel PPT-deleted vector to further reduce the low risk of insertional mutagenesis associated with systemic administration of IDLV's, b) to develop and test a novel IDLV-based human factor IX (hFIX) expression cassette as a means to minimize IDLV vector load required to fully correct FIX deficiency in hemophilia B mice, and c) to establish a novel packaging cell line to facilitate production of mobilization resistant IDLV's carrying expression cassettes in opposite orientation to the vector's LTR's. The proposal comprises three specific aims.
In Aim 1 we will focus on characterizing the ability of novel PPT-deleted IDLV's with reduced illegitimate integration to mediate efficient hepatic gene delivery, in vivo.
Aim 2 will focus on the development and testing of a novel hFIX cDNA with prolonged in vivo half-life (t1/2) and enhanced specific activity using IDLV's for hepatic gene delivery. The efficacy of the new IDLV's to correct FIX deficiency will be tested in hemophilia B mice. The focus of Aim 3 will be establishing of a novel RNA-regulated protein kinase (PKR) resistant packaging cell line generating high titers of gp64- pseudotyped vectors. A PPT-deletion will render the cell line-generated IDLV's less likely to illegitimately integrate, and internal expression cassettes incorporated in opposite orientation to the LTR's will render them less likely to be mobilized. Overall the proposed studies will yield a highly efficient and significantly safer gene delivery system most suitable for gene replacement therapy of nonfatal human diseases.

Public Health Relevance

Here we propose to employ two mechanistically independent approaches to minimize the levels of illegitimate integration in IDLV's. We will employ novel PPT-deleted IDLV's, which exhibit lower levels of illegitimate integration, and will develop a highly efficacious hFIX. In addition, we propose to establish a novel packaging cell line to produce high titer IDLV's carrying expression cassettes in opposite orientation to the LTR's.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Gene and Drug Delivery Systems Study Section (GDD)
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Eggerman, Thomas L
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University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
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